Bone graft technologies currently available to treat large segmental defects typically generate replacement tissue with poor vascularity and poor host integration that leads to clinical failures associated with osteonecrosis or dislodgement. As a result, there is a significant unmet need to improve the clinical outcome in procedures treating trauma, fracture non-unions, spinal fusion, osteonecrosis, and segmental bone osteotomies such as those created upon tumor removal. Tissue engineering is a promising strategy to promote bone regeneration. However, the general approach in bone tissue engineering to directly stimulate bone formation through osteogenesis has been largely unsuccessful. In this proposal we approach bone regeneration through a cartilage intermediate. This process, called endochondral ossification, is the normal developmental mechanism for formation of long bones, and is the pathway through which the majority of fractures heal. I hypothesize that bone regeneration through a chondrogenic intermediate will produce a neotissue that resembles the native bone in both form and function. To test this hypothesis I will compare bone regeneration from a cartilage graft to the gold-standard bone graft technique in a critically sized murine bone defect. In support of this approach, I have preliminary data demonstrating that a cartilage graft containing hypertrophic chondrocytes promotes a well-vascularized and integrated bone regenerate. In addition to evaluating the quality of bone regenerated by a cartilage graft versus bone graft, I will determine the mechanism through which repair occurs. According to my hypothesis, bone regeneration will occur through the process of endochondral ossification, resulting in apoptosis of hypertrophic chondrocytes and producing a bone regenerate that is host derived. However, preliminary data indicate that donor cartilage is contributing to the bone regenerate through an unresolved mechanism. I will use genetic and cell labeling techniques to trace the cell phenotype throughout this repair process to evaluate how the cartilage heals large bone defects. The second aim of this research proposal is to translate the concept of promoting bone regeneration through endochondral ossification into a clinically viable technology. To accomplish this I will design biologically modified synthetic scaffolds that promote formation of hypertrophic cartilage from mesenchymal stem cells (MSCs). Scaffold will be designed with variable degradation rates tuned to the process of endochondral ossification to optimize bone regeneration in vivo. In earlier studies I have characterized hypertrophic maturation of MSCs in tissue-engineered scaffolds and developed a MMP-7 bioresponsive system tuned to chondrogenesis. Together these aims address an important clinical problem with a translatable technology capable of improving current bone regeneration techniques. Furthermore this project was specifically designed to meet my long-term career objective related to musculoskeletal regeneration by utilizing multipotent progenitor cells and bioresponsive scaffolds to recapitulate normal development and/or repair mechanisms in clinically relevant in vivo models.